Cytomegalovirus assemblin: the amino and carboxyl domains of the proteinase form active enzyme when separately cloned and coexpressed in eukaryotic cells.
Cytomegalovirus assemblin: the amino and carboxyl domains of the proteinase form active enzyme when separately cloned and coexpressed in eukaryotic cells.
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巨细胞病毒组装蛋白：蛋白酶的氨基和羧基结构域在真核细胞中单独克隆和共表达时形成活性酶。
DOI：
10.1128/jvi.70.8.5395-5404.1996
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Gibson,W
中科院分区：
文献类型：
--
作者：
Hall,MR;Gibson,W
巨细胞病毒（CMV）丝氨酸蛋白酶组装素作为前体合成，经历三次主要的自身蛋白水解裂解。其中两个是所有疱疹病毒群的组装素同源物所共有的：一个在靠近前体羧基末端的成熟位点，另一个在靠近前体中点的释放位点。释放位点切割将蛋白水解氨基结构域，即组装素，从前体的非蛋白水解羧基结构域中解放出来。在CMV中，内部位点的第三个自蛋白水解裂解将组装素分成大小大致相同的氨基亚基（an）和羧基亚基（Ac），它们仍然作为活性“双链”酶结合在一起。我们克隆了编码An和Ac的序列，并分别用重组质粒转染人细胞和用重组杆状病毒感染昆虫细胞来表达它们。当来自猿猴CMV或人CMV的An和Ac在人或昆虫细胞中共表达时，形成了活性的双链组装素。这一发现表明，在这些真核生物系统中，An和Ac不需要作为单链组装素合成就可以折叠和正确结合，这表明它们可能在结构上（如果不是功能上）是不同的结构域。通过共免疫沉淀实验证明了独立表达的An和Ac亚基之间的相互作用，并且破坏复合物的努力表明亚基相互作用是疏水的。由独立表达的An和Ac形成的双链组装素的细胞切割实验也表明(i)其对CMV和单纯疱疹病毒原生底物的特异性与单链组装素相似，（ii） r位点切割对双链重组组装素的活性不是必需的，（iii）人类CMV和类人猿CMV的An和Ac重组亚基在功能上是可互换的。
The cytomegalovirus (CMV) serine proteinase assemblin is synthesized as a precursor that undergoes three principal autoproteolytic cleavages. Two of these are common to the assemblin homologs of all herpes group viruses: one at the maturational site near the carboxyl end of the precursor and another at the release site near the midpoint of the precursor. Release-site cleavage frees the proteolytic amino domain, assemblin, from the nonproteolytic carboxyl domain of the precursor. In CMV, a third autoproteolytic cleavage at an internal site divides assemblin into an amino subunit (An) and a carboxyl subunit (Ac) of approximately the same size that remain associated as an active "two-chain" enzyme. We have cloned the sequences encoding An and Ac as separate genes and expressed them by transfecting human cells with recombinant plasmids and by infecting insect cells with recombinant baculoviruses. When An and Ac from either simian CMV or human CMV were coexpressed in human or insect cells, active two-chain assemblin was formed. This finding demonstrates that An and Ac do not require synthesis as single-chain assemblin to fold and associate correctly in these eukaryotic systems, and it suggests that they may be structurally, if not functionally, distinct domains. An interaction between the independently expressed An and Ac subunits was demonstrated by coimmunoprecipitation experiments, and efforts to disrupt the complex indicate that the subunit interaction is hydrophobic. Cell-based cleavage assays of the two-chain assemblin formed from independently expressed An and Ac also indicate that (i) its specificity for both CMV and herpes simplex virus native substrates is similar to that of single-chain assemblin, (ii) R-site cleavage is not essential for the activity of two-chain recombinant assemblin, and (iii) the human CMV and simian CMV An and Ac recombinant subunits are functionally interchangeable.